Cryptosporidium parvum is a primary cause of cryptosporidiosis, an infectious disease affecting humans, which can become chronic and life threatening in immunocompromised individuals. A current limitation to the understanding of Cryptosporidium pathogenicity is a poor knowledge of the variability of virulence genes, such as those involved in attachment and invasion of host cells. The objectives of this project are to determine a] the variability of C. parvum genes with a demonstrated attachment/invasion function and b] the population structure and attachment/invasion gene variability of C. parvum in a defined geographic area. These objectives will allow us to test our central hypothesis that human pathogenic C. parvum display sequence variability in key attachment/invasion genes. The rationale for these objectives is that the successful characterization of sequence variability in attachment/invasion genes will be critical to the understanding of pathogenicity, assessment of vaccine targets and development of diagnostics. Two studies will be undertaken. The aim of the first study is to determine the variability in C. parvum genes that are necessary for attachment and invasion of host cells. The approach that will be used to achieve this aim is to identify and sequence polymorphic regions within key TRAP family genes from geographically diverse C. parvum isolates, and subject the alleles to codon based tests for selection. The second study aims to determine the population structure and extent of attachment/invasion gene variability among C. parvum isolates from human clinical infections in North Dakota, Minnesota and Wisconsin. A multilocus typing approach targeting 3 micro- and 3 minisatellite loci will be employed to compute evolutionary distances and determine the population structure of clinical C. parvum isolates. Attachment/invasion genes will be sequenced to determine the extent of their variability within the confines of the study area. The collective outcome from the proposed studies will address a critical gap in knowledge regarding C. parvum virulence genotypes. The significance of this outcome is its potential to advance a number of public health disciplines, including diagnostics and vaccine development, thereby contributing to the long term goal of reducing the incidence of human cryptosporidiosis. [unreadable] [unreadable] [unreadable]